The present invention relates to toll-like receptor 7 (TLR7) and toll-like receptor 8 (TLR8) agonists. More particularly, the present invention relates to compounds useful as agonists for TLR7 and/or TLR8, compounds useful as adjuvants, methods of making such compounds, pharmaceutical formulations comprising such compounds, and therapeutic use of the same.
The refinement and simplification of microbial vaccines and the use of synthetic and recombinant subunit antigens to improve vaccine manufacturability and safety has resulted in a decrease in vaccine potency. This has led to studies on the co-administration of adjuvants with antigens to potentiate vaccine activity and the weak immunogenicity of synthetic and recombinant epitopes. Adjuvants are additives that enhance humoral and/or cell mediated immune responses to a vaccine antigen. The design of vaccine adjuvants, however, has historically been difficult because of the complex nature of the molecular mechanisms involved in immune system function. Although the addition of microbial components has long been known to enhance adaptive immune responses, only recently was it shown that toll-like receptors (TLRs) on cells involved in immune surveillance, such as epithelial and dendritic cells, engage many of these microbial products via so-called “pathogen-associated patterns” or PAMPs. Many vaccine adjuvants and stand-alone immunomodulators appear to interact with members of the TLR family.
Of the ten known TLRs that have been identified in humans, five are associated with the recognition of bacterial components (TLRs 1, 2, 4, 5, 6) and four others (TLRs 3, 7, 8, 9) appear to be restricted to cytoplasmic compartments and are involved in the detection of viral RNA (TLRs 3, 7, 8) and unmethylated DNA (TLR9) (Iwasaki, A., Nat Immunol 2004, 5, 987). Activation of TLRs regulates intracellular signaling pathways and leads to gene expression via interaction with intracellular adapter molecules such as MyD88, TRIF, TIRAP, and TRAM (Akira, S. Nat Rev Immunol 2004, 4, 499; Takeda, K. Semin Immunol 2004, 16, 3). These adapter molecules can differentially regulate the expression of inflammatory cytokines/chemokines and type I interferons (IFN-α/β), which can lead to the preferential enhancement of antigen-specific humoral and cell-mediated immune responses (Zughaier, S. Infect Immun 2005, 73, 2940). Humoral immunity is the major line of defense against bacterial pathogens, whereas the induction of cytotoxic T lymphocytes (CTLs) appears to be crucial for protective immunity in the case of viral disease and cancer.
Currently, a group of aluminum salts known as alum are the dominant adjuvants used in human vaccines. But alum typically only enhances humoral (Th2) immunity and is generally used intramuscularly due to local toxicity by other routes (e.g., subcutaneous or intradermal inoculation leads to granulomas) (Aguilar, J. Vaccine 2007, 25, 3752). Other potential side effects of alum include increased IgE production, allergenicity and neurotoxicity. Thus, new safe and effective vaccine adjuvants are needed which are able to stimulate both antibody and Th1-type immune responses and that are compatible with different routes of administration and antigen formulations.
In the case of TLR7 and TLR8 activation, a few different classes of small molecule mimetics of the natural (U- and/or G-rich) viral ssRNA ligands have been identified. These include certain antiviral compounds related to oxidized guanosine metabolites (oxoguanosines), which primarily interact with TLR7 (Heil, F. Eur J Immunol 2003, 33, 2987; Hemmi, 2002) and derivatives of adenine which engage TLR7 and/or TLR8. The immune stimulating ability of these compounds has been attributed to the TLR/MyD88-dependent signaling pathways and the production of cytokines, including IL-6 and type I (particularly interferon-α) and type II interferons. TLR7 or TLR8 activation leads to the upregulation of co-stimulatory molecules (e.g. CD-40, CD-80, CD-86) and class I and II MHC molecules on dendritic cells (DCs). DCs are the principal cells of the immune system involved in uptake and presentation of antigens to T lymphocytes. Plasmacytoid dendritic cells (pDCs), which preferentially express TLR7, are professional interferon-α producing cells; whereas myeloid dendritic cells (mDCs) express TLR8. TLR8 activation on mDCs leads to the preferential production of pro-inflammatory cytokines such as IL-12, TNF-α, and IFN-γ and cell-mediated immunity (CMI).
One class of adenine derivatives that has received a considerable amount of attention are the 1H-imidazo[4,5-c]quinolines (IQs). The prototypical member of this class imiquimod (R847, S-26398) was found to be effective against genital papilloma virus infections, actinic keratosis, and basal cell carcinoma when applied topically in cream form. However, imiquimod has relatively low interferon-inducing activity and both oral and topical preparations are not without side-effects. In fact, serious side effects were reported in an hepatitis C virus (HCV) clinical trial with imiquimod. The large immunological “footprint” of TLR7 agonists in general has led concerns over toxicity. Clinical trials with another TLR7 agonist ANA-975, an oxoguanosine derivative, were recently suspended due to toxicity issues.
As a first-line therapy for hepatitis C virus (HCV) disease, interferon combinations can be highly effective at reducing viral load and in some subjects in eliminating viral replication. However, many patients fail to show a sustained viral response and in these patients viral load is not controlled. Additionally, therapy with injected interferon may be associated with a number of unwanted adverse effects which are shown to affect compliance (Dudley T, O'Donnell K, Haydon G, Mutimer D. Gut., 2006, 55(9):1362-3). Administration of a small molecule compound which could stimulate the innate immune response, including the activation of type I interferons and other cytokines, could become an important strategy for the treatment or prevention of human diseases including viral infections. This type of immunomodulatory strategy has the potential to identify compounds which may be useful not only in infectious diseases but in cancer (Krieg. Curr. Oncol. Rep. 2004; 6(2):88-95), allergic diseases (Moisan et al., Am. J. Physiol. Lung Cell Mol. Physiol. 2005; 290(5):L987-95), other inflammatory conditions such as irritable bowel disease (Rakoff-Nahoum S., Cell. 2004, 23; 118(2):229-41), and as vaccine adjuvants (Persing et al. Trends Microbiol. 2002; 10(10 Suppl):S32-7).
Another member of the IQ class of TLR7/8 ligands and a derivative of a metabolite of imiquimod is resiquimod. Resiquimod (R-848, S-28609) also activates TLR7 in macrophages and DCs in a MyD88-dependent manner either directly or indirectly via an accessory molecule and upregulates co-stimulatory molecules and MHCI/II in DCs. But in contrast to imiquimod, the more potent and toxic resiquimod is also a ligand for TLR8 signaling, which leads to the reversal of CD4+ regulatory (Treg) cell function. Using transfected HEK293 cells, it was recently shown that TLR7 agonists are more effective at generating IFN-α and IFN-regulated cytokines, whereas TLR8 agonists were more effective at inducing proinflammatory cytokines such as TNF-α and IL-12, suggesting that TLR7 activation may be more important for antibody responses (Th2-type responses) while TLR8 activation could drive CMI or Th1-type immune responses. However, as mentioned above, many TLR7/8 agonists often display toxic properties, are unstable, and/or have unsubstantial immunostimulatory effects. Thus, the discovery and development of effective and safe adjuvants that activate TLR7 and/or TLR8 is essential for improving the efficacy and safety of existing and new vaccines via helping to control the magnitude, direction, and duration of the immune response against antigens.
Unlike TLR2 and TLR4, which recognize PAMPs on cell surfaces, TLR7/8 PAMPs are sensed in the endosomal/lysosomal compartments and require endosomal maturation. Cellular uptake is prerequisite for cellular activation in the case of natural and zenobiotic TLR7/8 ligands such as imiquimod and resiquimod. Thus, strategies that would increase the penetration of the TLR7/8 ligand into DCs and other immune cells could enhance TLR activation and vaccine efficacy as well as ameliorate toxic effects.
The immune stimulating ability of certain antiviral/antitumor 1H-imidazo[4,5-c]quinolines (Gerster, J. F, et al. J. Med. Chem. 2005, 48, 3481-3491), such as imiquimod, has been attributed primarily to the activation of TLR7 in plasmacytoid dendritic cells and B cells and the induction of type I interferons (IFNα/β) and IFN-regulated cytokines (Miller, R. L.; Meng, T.-C.; Tomai, M. A. Drug News Perspect. 2008, 21, 69-87.).
TLR7/8 agonists in which an imidazoquinoline is conjugated to a phospho- or phosphonolipid have enhanced immune responses. It has been suggested that these agonists could improve immune responses due to direct intereaction of these compounds with endosomal TLR7 and/or TLR7/8 and/or interaction of an active metabolite after enzymatic action. (WO2010/04850).